What are the intracellular signals for agonist‐activated calcium entry into hepatocytes?

The Ca 2+ signal observed in individual fura‐2‐loaded hepatocytes stimulated with the α 1 ‐adrenergic agonist phenylephrine consisted of a variable latency period, a rapid biphasic increase in the cytosolic free Ca 2+ , followed by a period of maintained elevated cytosolic Ca 2+ (plateau phase) that depended on the continued presence of both agonist and external Ca 2+ , Microinjection of guanosine‐5′‐ O ‐(3‐thiophosphate) elicited a Ca 2+ transient with the same basic features. The Ca 2+ transient resulting from microinjecting inositol 1,4,5‐trisphosphate (Ins‐1,4,5‐P 3 ) occurred with essentially no latency period and consisted of a rapid spike that decayed back to preinjection levels within 15 s. Microinjection of inositol 1,4‐5‐trisphosphorothioate (thio‐IP 3 ), a nonmetabolizable analog of Ins‐1,4,5‐P 3 , elicited a Ca 2+ transient that was initially identical to that observed with Ins‐1,4,5‐P 3 , except that the cytosolic Ca 2+ remained elevated. The maintained thio‐IP 3 ‐induced Ca 2+ increase was dependent on the presence of external Ca 2+ , suggesting an activation of Ca 2+ influx. Reintroduction of external Ca 2+ in the presence of 5 μM phenylephrine to Ca 2+ ‐depleted cells resulted in a 2‐fold greater rate of rise in the cytosolic Ca 2+ compared to the rate observed upon Ca 2+ addition to cells Ca 2+ ‐depleted by preatement with thapsigargin. The rate of Ca 2+ rise upon Ca 2+ addition to cells microinjected with thio‐IP 3 was similar to that observed with phenylephrine. Coinjection of the cells with thio‐IP 3 plus heparin reduced the rate of Ca 2+ rise upon Ca 2+ addition to that observed in thapsigargin‐treated cells. These data indicate that the mechanism responsible for receptor‐mediated stimulation of Ca 2+ entry into hepatocytes involves not only capacitative Ca 2+ entry but also an additional component mediated directly by Ins‐1,4,5‐P 3 .